Regulation Mechanism Of Wolbachia On The Detoxification Of Nilaparvata Lugens (st(?)l)

As the most insecticide-resistant grain crops pest,Nilaparvata lugens have experienced frequent resistance events in recent years.Enhanced detoxification metabolism is the main cause of insecticide resistance in N.lugens.Recent studies have shown that antibiotic stress can affect the expression of core detoxifying genes by disrupting the structure of N.lugens bacterial symbionts,thereby inhibiting the detoxification metabolism of the host.These results confirmed a potential mechanism of interaction between N.lugens bacterial symbionts and host detoxifying gene expression.Based on our previous study,insecticide stress significantly induced an increase in Wolbachia content in N.lugens,it was hypothesized that Wolbachia plays an active role in the detoxification metabolism of the host to insecticides.In this study,we screened and identified Wolbachia as a vital bacterial symbiont that regulates the detoxification metabolism of N.lugens,confirmed that Wolbachia mediates the variation of insecticide susceptibility to high temperature and tetracycline stress in the brown planthopper,clarified the effect of Wolbachia on host detoxification metabolism,and analyzed the signaling pathway of Wolbachia in response to insecticide regulation of host detoxification metabolism.The main results are as follows.1.Screening and identification of bacterial symbionts regulating the expression of N.lugens detoxifying genesThe antibiotics tetracycline,ciprofloxacin and sulbactam targeting different brown planthopper core microorganisms were screened based on the rice root dip method.The three antibiotics selectively inhibited the brown planthopper core bacterial symbionts,shaped the brown planthopper containing the three symbionts’ structures,and impact detoxifying gene expression profiles of N.lugens significantly.Tetracycline-shaped bacterial symbiont structure was significantly correlated with detoxifying gene profile based on Procrustes analysis.Wolbachia,a symbiont that was specifically inhibited by tetracycline,and its content were significantly and positively correlated with the expression of Nl CYP4CE1 and Nl CYP6CW1,the core detoxifying P450 gene of N.lugens,with a correlation coefficient of 0.76 and-0.46,indicating that Wolbachia plays a vital role in the expression of detoxification metabolism genes of N.lugens.2.Wolbachia mediates insecticide susceptibility variation under high temperature and tetracycline stress in N.lugensTo further validate the relationship between Wolbachia and host detoxifying gene expression,the structure of N.lugens bacterial symbionts was disrupted based on tetracycline and high temperature stresses and successfully suppressed Wolbachia,the results demonstrated that the susceptibility of N.lugens to insecticide was significantly increased in Wolbachia suppressed strains with the activities of P450 and GST were significantly decreased.In these two treatments,30(4 GST and 25 P450 genes)and 20(7GST and 13 P450 genes)detoxifying genes were significantly down-regulated,respectively,of which 11(1 GST and 10 P450 genes)genes were significantly downregulated in both two treatments,including the core P450 genes Nl CYP4CE1 and Nl CYP6AY1 which indicated that these two genes play a key role in susceptibility variations in high temperature and tetracycline treated N.lugens strains.The high temperature had no significant effect on the detoxification metabolism of tetracycline-treated N.lugens.These results revealed that the variation of N.lugens insecticide susceptibility under high temperature and tetracycline stress depended on bacterial symbionts,especially Wolbachia.It further mediated host detoxification metabolism to insecticides by enhancing detoxification enzyme activity through regulating host detoxifying gene expression.3.Wolbachia mediated N.lugens and Drosophila melanogaster detoxification metabolism was clarifiedTo clarify the influence of Wolbachia on the detoxification of N.lugens and D.melanogaster.The effect of Wolbachia infection on host insecticide detoxification metabolism was analyzed based on the beneficent WI and WU and the D.melanogaster lines Dmel w Mel and Dmel w Mel T.The results showed that Wolbachia infection significantly reduced susceptibility to insecticides,increased host detoxification enzymes P450 and GST and increase the expression of the core detoxifying gene of N.lugens.Especially,Wolbachia induced up-regulation of imidacloprid metabolism-related P450 gene expression.Similarly,Wolbachia significantly enhanced insecticide resistance of D.melanogaster by increasing imidacloprid metabolism-related gene Dm CYP6g1 and Dm CYP6g2,indicating that Wolbachia mediated the host detoxification metabolism by regulating the expression of P450 gene.4.Dissection of the signaling pathway of Wolbachia in response to insecticide regulation of host detoxification metabolismTo clarify the mechanism of Wolbachia regulation detoxifying gene of N.lugens,A core effector,Nl Cnc C between Wolbachia and host detoxification metabolism genes was screened based on analyzed the expression of xenobiotic receptor family transcription factor.Silencing Nl Cnc C significantly inhibits the expression of host detoxifying genes,especially the core P450 genes Nl CYP4CE1 and Nl CYP6AY1,thereby reducing detoxifying enzyme activity and increasing susceptibility of N.lugens to insecticide.In addition,silencing of Nl Cnc C blocked the effect of high temperature on N.lugens insecticide susceptibility but did not affect the inhibition of bacterial symbionts content.These results indicate that the Nl Cnc C pathway mediates Wolbachia regulated host detoxification.To elucidate the mechanism of Wolbachia regulation of host detoxification metabolism in response to insecticides,we analyzed the Wolbachia content and the expression of the immune-related gene Nl Duox in N.lugens by insecticide stress.The results found that complementary trends between Nl Duox and Wolbachia content.Nl Duoxdependent ROS was significantly affecting the content of Wolbachia were confirmed with RNAi and paraquat activation.However,silencing Nl Duox significantly inhibited the expression of Nl CYP4CE1 and Nl CYP6ER1.It was revealed that Nl Duox-dependent ROS with Wolbachia synergistically activated the sustained expression of core P450 genes of the host to metabolize insecticides.In conclusion,this study elucidated the mechanism by which Wolbachia regulates the detoxification metabolism of the insect host,N.lugens.The results of this study help to deepen the understanding of the theory of insect host-symbionts interactions.